As it has become increasingly apparent that gene expression in individual cells deviates significantly from the average behavior of cell populations, new methods that provide accurate integer counts of mRNA copy numbers in individual cells are needed. Ideally, such methods should also reveal the intracellular locations of the mRNAs, as mRNA localization is often used by cells to spatially restrict the activity gene.
In situ hybridization, followed by microscopic analysis, is a well-established means of studying gene expression. The first generation of in situ hybridizations was performed with radioactive probes. Early improvements involved linking the probes to enzymes that catalyze chromogenic or fluorogenic reactions. However, because the products of these reactions were small molecules or precipitates that diffuse away from the probe, the location of the target molecules could not be precisely determined. Conversely, probes labeled directly with a few fluorophores maintained spatial resolution, but the sensitivity that can be achieved is relatively poor.
Robert Singer and colleagues developed an in situ hybridization procedure that was not only sensitive enough to permit the detection of single mRNA molecules, but also restricted the signals to close proximity of the targets. They hybridized five oligonucleotide probes simultaneously to each mRNA target, each of which was about 50-nucleotides in length and each of which was labeled with five fluorophore moieties. Although the authors convincingly demonstrated single molecule sensitivity and other groups have successfully used these probes, the system has not been widely adopted. One reason for this is difficulty in the synthesis and purification of heavily labeled oligonucleotides. Usually, flurophore moieties are introduced via primary amino groups that are incorporated into oligonucleotides during their synthesis. When multiple amino groups are introduced into the same oligonucleotide some are lost due to side reactions such as transamidation. Coupling of fluorophores to the remaining amino groups is inefficient and requires several consecutive coupling reactions and it is difficult to purify oligonucleotides in which all designed sites are coupled to fluorophores from those that are partially coupled. Also, when some fluorophores are present in multiple copies on the same oligonucleotide they interact with each other altering the hybridization characteristics of the oligonucleotides and exhibiting severe self-quenching. These problems are obviated if each probe had just a single terminal amino group to serve as the site of attachment.
Another issue with the use of small numbers of heavily labeled probes is that a significant portion of the fluorescence is lost for every probe that does not bind to the target, whereas every non-specific binding event increases the background. This leads to a widened distribution of number of probes bound to each target mRNA. For instance, when using 5 fluorescent probes targeted to a single mRNA, Femino et al estimated that the majority of the fluorescent spots observed had intensities indicating the presence of only 1 or 2 probes. Science 280, 585-590 (1998). This makes it difficult to unambiguously identify those fluorescent spots as mRNA molecules, since it is impossible to determine whether the detection of an individual probe arises from legitimate binding to the target mRNA or non-specific binding. These “thresholding” problems limit the ability of such methods to provide reliable counts of mRNA numbers in individual cells.
Thus there remains a need for improved methods to provide reliable counts of mRNA numbers in individual cells and a need for probes that are easily synthesized and purified.